U.S. patent application number 11/937924 was filed with the patent office on 2009-05-28 for benzenesulfonic acid salt compounds.
This patent application is currently assigned to Tanabe Seiyaku Co.,Ltd.. Invention is credited to Masataka HIKOTA, Toru Iijima, Michael S. McClure.
Application Number | 20090137658 11/937924 |
Document ID | / |
Family ID | 40670281 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090137658 |
Kind Code |
A1 |
HIKOTA; Masataka ; et
al. |
May 28, 2009 |
BENZENESULFONIC ACID SALT COMPOUNDS
Abstract
Besylate salts of
trans-4-({2-[(2S)-2-cyano-pyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyc-
lohexanecarboxamide are described as well as methods of using the
same in the treatment of disorders characterized by
hyperglycemia.
Inventors: |
HIKOTA; Masataka;
(Shiki-shi, JP) ; Iijima; Toru; (Toda-shi, JP)
; McClure; Michael S.; (Cary, NC) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Tanabe Seiyaku Co.,Ltd.
|
Family ID: |
40670281 |
Appl. No.: |
11/937924 |
Filed: |
November 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10837579 |
May 4, 2004 |
|
|
|
11937924 |
|
|
|
|
60468329 |
May 6, 2003 |
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Current U.S.
Class: |
514/423 ;
548/540 |
Current CPC
Class: |
C07D 207/16
20130101 |
Class at
Publication: |
514/423 ;
548/540 |
International
Class: |
A61K 31/40 20060101
A61K031/40; C07D 295/182 20060101 C07D295/182 |
Claims
1-22. (canceled)
23. A compound comprising a crystalline form of a benzenesulfonic
acid salt of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino-N,N-dimet-
hylcyclohexanecarboxamide.
24. The compound of claim 23, wherein said crystalline form of a
benzene sulfonic acid salt is an anhydrate.
25. The compound of claim 24, wherein said crystalline form of a
benzenesulfonic acid salt exhibits a melting point of 157.degree.
C.
26. The compound of claim 24, wherein said crystalline form of a
benzenesulfonic acid salt is obtainable by the reaction of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2oxoethyl}-amino)-N,N-dimethylcycl-
ohexanecarboxamide with benzene sulfonic acid and crystallization
in a polar solvent.
27. A compound, comprising a crystalline form of an anhydrous
besylate salt of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dime-
thylcyclohexanecarboxamide, wherein said crystalline form exhibits
a powder x-ray diffraction pattern comprising the following peaks:
TABLE-US-00007 Two theta (deg) d-spacing (angstroms) 6.1 .+-. 0.2
14.5 .+-. 0.5 14.1 .+-. 0.2 6.3 .+-. 0.1 16.3 .+-. 0.2 5.4 .+-. 0.1
19.8 .+-. 0.2 4.5 .+-. 0.1 22.4 .+-. 0.2 4.0 .+-. 0.1
28. The compound of claim 23, wherein said crystalline form of a
benzenesulfonic acid salt is a monohydrate.
29. The compound of claim 28, wherein said crystalline form of a
benzenesulfonic acid salt exhibits a melting point ranging from 110
to 120.degree. C.
30. The compound of claim 28, wherein said crystalline form of a
benzene sulfonic acid salt is obtainable by the reaction of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide with benzene sulfonic acid and crystallization
in a mixture of a polar solvent and water.
31. A compound comprising a crystalline form of a monohydrate
besylate salt of the compound,
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide, wherein said crystalline form exhibits a powder
x-ray diffraction pattern comprising the following peaks:
TABLE-US-00008 Two theta (deg) d-spacing (angstroms) 6.8 .+-. 0.2
13.0 .+-. 0.4 7.8 .+-. 0.2 11.3 .+-. 0.3 8.8 .+-. 0.2 10.0 .+-. 0.3
17.4 .+-. 0.2 5.1 .+-. 0.1 26.7 .+-. 0.2 3.3 .+-. 0.1
32. A process for preparing the compound of claim 23 or 24,
comprising forming said crystalline form of a benzenesulfonic acid
salt by reacting
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide with benzenesulfonic acid, followed by
crystallization in a polar solvent.
33. A process for preparing the compound of claims 23 or 28,
comprising forming said benzenesulfonic acid salt by reaction of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide with benzenesulfonic acid, followed by
crystallization in a mixture of a polar solvent and water.
34. A pharmaceutical composition, comprising: a therapeutically
effective amount of a compound according to any one of claims 23-31
or 39, and one or more of pharmaceutically acceptable carriers,
diluents and excipients.
35. A pharmaceutical composition comprising: a crystalline form of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate anhydrate according to claim 27; or a
crystalline of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate monohydrate according to claim 31; or a
mixture thereof.
36. The pharmaceutical composition of claim 34, comprising at least
one hydrate of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate.
37. The pharmaceutical composition of claim 34, further comprising
at least one solvate of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate.
38. The compound of any one of claims 23-31 or 39, for use in
medical therapy.
39. The compound of claim 23, wherein said crystalline form of a
benzenesulfonic acid salt is a crystalline anhydrate, hydrate, or
solvate.
Description
[0001] This is a continuation of U.S. application Ser. No.
10/837,579, filed May 4, 2004, now abandoned, which claims the
benefit of U.S. provisional application No. 60/468,329, filed May
6, 2008, the contents of all of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to anhydrate and monohydrate
benzenesulfonic acid salts. In particular, to the anhydrate and
monohydrate forms of the benzenesulfonic acid salt of
(2S)-2-cyanopyrrolidinyl-2-oxoethyl amine derivatives. These
compounds are inhibitors of serine proteases, such as dipeptidyl
peptidases, and are useful in the treatment of disorders such as
hyperglycemia and/or other conditions of diabetes. The particular
forms disclosed herein demonstrate unexpectedly beneficial physical
properties for use as commercial medicaments.
[0004] 2. Description of the Related Art
[0005] International Patent Application PCT/JP01/08803 filed May
10, 2001, and published as WO02/30891 on 18/Apr./2002, discusses
inhibitors of serine proteases including Dipeptidyl Peptidase IV
(DPP IV), and discloses aliphatic nitrogen-containing 5-membered
ring compounds which demonstrate such activity, including
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide. However, several salts of these
compounds have been found to absorb very large amounts of water at
the expected exposure humidities if utilized in a medicament (e.g.,
20-75% relative humidity (RH)). As a result, suitability of the
compound as a medicament could be compromised unless there were
special handling and storage procedures instituted.
SUMMARY OF THE INVENTION
[0006] The present inventors have now identified novel
benzenesulfonic acid salts of (2S)-2-cyanopyrrolidinyl-2-oxoethyl
amines, which are suitable as serine protease inhibitors. These
benzenesulfonic acid salts have moisture sorption properties
superior to the HCl salts of (2S)-2-cyanopyrrolidinyl-2-oxoethyl
amines disclosed in the art. The compounds may be prepared in
crystal form and therefore have good physical stability. That is,
the benzenesulfonic acid salts of the present invention sorb much
lower amounts of water when exposed to a broad range of humidities
and can be prepared in a physically stable crystal form, thus
enhancing their suitability as medicaments.
[0007] In a first aspect of the present invention, there is
provided a compound of Formula I,
##STR00001##
and anhydrate, hydrate or solvate forms thereof.
[0008] In one embodiment, the present invention provides a
crystalline form of anhydrous
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate characterised by a powder
x-ray diffraction pattern comprising the following peaks:
TABLE-US-00001 Two theta (deg) d-spacing (angstroms) 6.1 .+-. 0.2
14.5 .+-. 0.5 14.1 .+-. 0.2 6.3 .+-. 0.1 16.3 .+-. 0.2 5.4 .+-.
0.1
[0009] More particularly, the crystalline form has a powder x-ray
diffraction pattern that is substantially as shown in FIG. 1. The
relative intensities of the diffraction peaks can vary due to
variation in the preferred orientation of particles in the
diffraction experiment sample preparation. The observed relative
intensities are also influenced by the specific instrumental
geometry of the powder diffractometer.
[0010] In another embodiment, the present invention provides a
crystalline form of monohydrate
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate characterised by a powder
x-ray diffraction pattern comprising the following peaks:
TABLE-US-00002 Two theta (deg) d-spacing (angstroms) 6.8 .+-. 0.2
13.0 .+-. 0.4 7.8 .+-. 0.2 11.3 .+-. 0.3 8.8 .+-. 0.2 10.0 .+-.
0.3
[0011] More particularly, the crystalline form has a powder x-ray
diffraction pattern that is substantially as shown in FIG. 2. The
relative intensities of the diffraction peaks can vary due to
variation in the preferred orientation of particles in the
diffraction experiment sample preparation. The observed relative
intensities are also influenced by the specific instrumental
geometry of the powder diffractometer.
[0012] In a second aspect of the present invention, there is
provided a pharmaceutical composition including a therapeutically
effective amount of a compound of Formula I and anhydrate, hydrate
or solvate forms thereof.
[0013] Furthermore, the present invention should be interpreted to
include pharmaceutical compositions that include one or more
anhydrate form of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate, one or more hydrated form of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate, and/or one or more solvated
form of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate.
[0014] Preferably, as used herein pharmaceutical compositions
include one or more pharmaceutically acceptable carrier, diluent,
or excipient.
[0015] In a third aspect of the present invention, there is
provided a method of treating a disorder in a mammal having
hyperglycemia or other conditions associated with diabetes,
including: administering to said mammal a therapeutically effective
amount of a compound of Formula I, and anhydrate, hydrate or
solvate forms thereof.
[0016] In a fourth aspect of the present invention, there is
provided a compound of Formula I and anhydrate, hydrate or solvate
forms thereof, for use in medical therapy.
[0017] In a fifth aspect of the present invention, there is
provided use of a compound of Formula I and anhydrate, hydrate or
solvate forms thereof, in the preparation of a medicament for use
in the treatment and/or prophylaxis of hyperglycemia or other
conditions associated with diabetes.
[0018] It will be appreciated by the skilled person that a
`benzenesulfonic acid` salt may also be referred to as a
`benzenesulfonate` or `besylate` salt.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 depicts the powder X-ray diffraction pattern (PXRD)
of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}-amino)-N,N-dimethylcyc-
lohexanecarboxamide besylate anhydrate, using a conventional powder
X-ray diffractometer with Bragg-Brentano geometry and copper K
alpha radiation.
[0020] FIG. 2 depicts the powder X-ray diffraction pattern (PXRD)
of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}-amino)-N,N-dimethylcyc-
lohexanecarboxamide besylate monohydrate, using a conventional
powder X-ray diffractometer with Bragg-Brentano geometry and copper
K alpha radiation.
[0021] FIG. 2a depicts the simulated powder X-ray diffraction
pattern (PXRD) of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-di-
methylcyclohexanecarboxamide besylate monohydrate with copper
K-alpha radiation.
[0022] FIGS. 3 (a) and (b) depict water sorption curves of (a)
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate monohydrate and (b) trans
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}-amino)-N,N-dimethylcyclohexa-
necarboxamide besylate anhydrate.
[0023] FIG. 4 depicts infrared (IR) studies relating to
hydration/dehydration of
trans-4-({2-[(2S)-2-cyano-pyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyc-
lohexanecarboxamide besylate monohydrate.
[0024] FIG. 5 depicts the Raman spectra of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate monohydrate.
[0025] FIG. 6 depicts the Raman spectra of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate anhydrate.
[0026] FIG. 7 depicts the Raman spectra of the ethanol solvate of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate.
[0027] FIG. 8 depicts the Raman spectra of the 1-propanol solvate
of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}-amino)-N,N-dimethylcyc-
lohexanecarboxamide besylate.
[0028] FIG. 9 depicts the Raman spectra of the 2-propanol solvate
of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}-amino)-N,N-dimethylcyc-
lohexanecarboxamide besylate.
[0029] FIG. 10 depicts the Raman spectra of the 2-methyl-1-propanol
solvate of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate.
[0030] FIG. 11 depicts the Raman spectra of the acetone solvate of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate.
[0031] FIG. 12 depicts the powder X-ray diffraction pattern (PXRD)
of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}-amino)-N,N-dimethylcyc-
lohexanecarboxamide hydrochloride.
DETAILED DESCRIPTION OF THE INVENTION
[0032] As discussed and illustrated throughout, the present
invention includes certain solid state crystalline forms. Several
methods for characterizing such forms exist, and the invention
should not be limited by the methods chosen or the instrumentation
used in characterizing the compounds of the present invention. For
example, with regard to x-ray diffraction patterns, the diffraction
peak intensities in the experimental patterns can vary, as is known
in the art, primarily due to preferred orientation (non-random
orientation of the crystals) in the prepared sample. As such, the
scope of the present invention must be considered in light of the
variability of characterization that is appreciated by those
skilled in the art.
[0033] As used herein, the term "effective amount" means that
amount of a drug or pharmaceutical agent that will elicit the
biological or medical response of a tissue, system, animal or human
that is being sought, for instance, by a researcher or clinician.
Furthermore, the term "therapeutically effective amount" means any
amount which, as compared to a corresponding subject who has not
received such amount, results in improved treatment, healing,
prevention, or amelioration of a disease, disorder, or side effect,
or a decrease in the rate of advancement of a disease or disorder.
The term also includes within its scope amounts effective to
enhance normal physiological function.
[0034] As used herein, the terms "anhydrous" and "anhydrate" are
used interchangeably. Likewise the terms "hydrous" and "hydrate"
are used interchangeably.
[0035] Novel benzenesulfonic acid salts of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide, which are suitable as serine protease
inhibitors, have moisture sorption properties superior to other
crystalline salts of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxo-ethyl}amino)-N,N-dimethylcyc-
lohexanecarboxamide disclosed in the art. This is exemplified by
the hygroscopicity of the crystalline hydrochloride (HCl) salt of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide. The crystalline HCl salt of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide has been observed to deliquesce at
25.degree. C. at relative humidities greater than 75% in
approximately 20 hours. Additionally, this crystalline HCl salt was
found to deliquesce at 40.degree. C. at relative humidities of
greater than 75% in approximately 24 hours. Comparatively, the
benzenesulfonic acid salts of
trans-4-({2-[(2S)-2-cyano-pyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyc-
lohexanecarboxamide have not been observed to deliquesce under
these same conditions. That is, the benzenesulfonic acid salts of
the present invention sorb much lower amounts of water when exposed
to a broad range of humidities and can be prepared in a physically
stable crystal form, thus enhancing their suitability as
medicaments.
[0036] Benzenesulfonic acids of Formula I have been observed to
exist in a monohydrate form, designated Form 1, and an anhydrate
form, designated Form 2. The monohydrate of the compound of Formula
I, Form 1, has a theoretical water content of 3.73% w/w.
Additionally, the monohydrate sorbs another 1-2% w/w water up to
95% relative humidity. Infrared (IR) studies have shown that the
monohydrate loses water readily at room temperature when purged
with dried air or nitrogen and re-hydration occurs rapidly upon
exposure of the dehydrated form to the atmospheric humidity. It has
also been observed that the anhydrate form converts to the hydrate
in the presence of water vapour, such as at elevated relative
humidity.
[0037] Additional infrared (IR) studies (FIG. 4) have shown that
when the monohydrate Form 1 of Formula I is exposed to dry nitrogen
for a given period of time, partial dehydration of the hydrate is
facile and can occur under these low relative humidity conditions
at room temperature. When exposed to higher relative humidities,
re-hydration to the monohydrate occurs very rapidly. The partially
dehydrated form of the monohydrate Form 1 of Formula I is stable
for at least 24 hours at room temperature and partial hydration to
the monohydrate of Formula I can be recovered at higher relative
humidity (RH).
[0038] Importantly, although the above-referenced water contents
are noted, the water content should not be considered as
descriptive of any particular pharmaceutical composition or
formulation comprising the forms of the present invention. Rather,
when in admixture with other pharmaceutically acceptable carriers,
diluents, or excipients, the water content may be higher or lower.
The water contents given above should be considered as descriptive
of the specific forms, themselves.
[0039] As examples of the preferred compounds of the present
invention, anhydrous Form 2 may be characterized by, among other
properties, a melting point of about 157.degree. C. Likewise
monohydrate Form 1 may be characterized by, among other properties,
a melting point which occurs between 110 to 120.degree. C.
[0040] In one embodiment, the compound is the anhydrate Form 2 of
the compound of Formula I characterized, in part, by a powder x-ray
diffraction pattern as shown in FIG. 1. The anhydrate Form 2 of the
compound of Formula I may be characterised by including, but not
limited to, the peaks of Table I.
TABLE-US-00003 TABLE I (Form 2 anhydrate) Two theta (deg) *
d-spacing (angstroms) 6.1 .+-. 0.2 14.5 .+-. 0.5 14.1 .+-. 0.2 6.3
.+-. 0.1 16.3 .+-. 0.2 5.4 .+-. 0.1 19.8 .+-. 0.2 4.5 .+-. 0.1 22.4
.+-. 0.2 4.0 .+-. 0.1 * Based on Cu K.alpha. radiation. K.alpha.2
was removed prior to peak location
[0041] Notably, in a mixture of the anhydrate Form 2 of the
compound of Formula I with another phase, not all the peaks listed
in Table I may be apparent in the mixture's powder diffraction
pattern.
[0042] In another embodiment, the compound is the monohydrate Form
1 of the compound of Formula I characterized, in part, by a powder
x-ray diffraction pattern as shown in FIG. 2. The monohydrate Form
1 of the compound of Formula I may be characterised by including,
but not limited to, the peaks of Table II.
TABLE-US-00004 TABLE II (Form 1 monohydrate) Two theta (deg) *
d-spacing (angstroms) 6.8 .+-. 0.2 13.0 .+-. 0.4 7.8 .+-. 0.2 11.3
.+-. 0.3 8.8 .+-. 0.2 10.0 .+-. 0.3 17.4 .+-. 0.2 5.1 .+-. 0.1 26.7
.+-. 0.2 3.3 .+-. 0.1 * Based on Cu K.alpha. radiation. K.alpha.2
was removed prior to peak location
[0043] Notably, in a mixture of the monohydrate Form 1 of the
compound of Formula I with another phase, not all the peaks listed
in Table II may be apparent in the mixture's powder diffraction
pattern.
[0044] The compounds of Formula I include within their scope
substantially pure anhydrate, hydrate or solvate forms, as well as
mixtures of solvate, hydrate and anhydrate forms. It is also
understood, that such compounds include crystalline or amorphous
forms and mixtures of crystalline and amorphous forms. The term
`substantially pure` means less than 10% of another form,
preferably less than 5%, more preferably less than 1%, is
present.
[0045] The free base and HCl salts of the compounds of Formula I
may be prepared according to the procedures of the International
Patent Application publication WO02/30891, referred to above.
[0046] As illustrated in Scheme A, the compound of Formula I, i.e.,
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate has been prepared in two
distinct forms, a monohydrate form (Form 1) (Formula I' in Scheme
A) and an anhydrate form (Form 2) (Formula I'' in Scheme A). The
relationship of these forms is illustrated in Scheme B below.
Anhydrate Form 2
[0047] The anhydrate Form 2 of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate may be prepared by (a)
reacting (2S)-1-(chloroacetyl)-2-pyrrolidinecarbonitrile with
trans-4-amino-N,N-dimethylcyclohexane-carboxamide in acetonitrile
in the presence of a base, such as potassium carbonate, followed by
(b) introducing a dilute acid solution, such as a solution of
citric acid and an extraction solvent such as dichloromethane and
separating the layers, (c) adding 5N sodium hydroxide to adjust the
pH to within a range of 8-11 to the aqueous layer and adding an
extraction solvent such as dichloromethane, (d) separating the
organic phase, and then (e) solvent exchanging the dichloromethane
for a higher boiling temperature solvent such as methyl acetate (f)
adding benzenesulfonic acid hydrate to the solution (g) and
distilling the azeotrope of methyl acetate, for example, and water
to provide the besylate anhydrate. Interconversion to the
monohydrate and back to the anhydrate of the besylate salt
compounds of the invention is as depicted in Scheme B.
Monohydrate Form 1
[0048] The monohydrate Form 1 of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate may be prepared by (a)
reacting (2S)-1-(chloroacetyl)-2-pyrrolidinecarbonitrile with
trans-4-amino-N,N-dimethylcyclohexane-carboxamide in acetonitrile
in the presence of a base, such as potassium carbonate, followed by
(b) introducing an dilute acid solution, such as a solution of
citric acid and an extraction solvent such as dichloromethane and
separating the layers, (c) adding 5N sodium hydroxide to adjust the
pH to within a range of 8-11 to the aqueous layer and adding an
extraction solvent such as dichloromethane, (d) separating the
organic phase, and then (e) solvent exchanging the dichloromethane
for a higher boiling temperature solvent such as 2-butanone (f)
adding water to the 2-butanone solution (g) and adding
benzenesulfonic acid hydrate to the solution to provide the
besylate monohydrate.
[0049] Thus, the anhydrate form of the compound of formula I is
obtainable by reaction of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide with benzenesulphonic acid and
crystallisation in a polar solvent, e.g. methyl acetate (preferably
anhydrous methyl acetate), and the monohydrate form of the compound
of formula I is obtainable by reaction of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide with benzenesulphonic acid and
crystallisation in a mixture of a polar solvent, e.g. 2-butanone,
and water.
[0050] In another aspect, the present invention provides a process
for preparing a compound of Formula I by reaction of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide with benzenesulfonic acid followed by
crystallisation in a polar solvent, e.g. methyl acetate.
[0051] In another aspect, the present invention provides a process
for preparing a compound of Formula I by reaction of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide with benzenesulfonic acid followed by
crystallisation in a mixture of a polar solvent, e.g. 2-butanone,
and water.
##STR00002##
##STR00003##
[0052] A series of crystallization experiments were performed to
investigate whether the benzenesulfonic acid salt of Formula I can
either exist in more than one solid-state form or has a propensity
to form solvates. This series of experiments employed 45 different
solvent systems and four crystallization modes (slow evaporation,
fast evaporation, cooling, and ripening). These experiments
indicated the existence of at least two non-solvated solid-state
forms, the monohydrate and the anhydrate of the compound of Formula
I. Additionally, benzenesulfonic acid salts of Formula I were shown
to form solvates in the presence of several hydrogen-bond-donor
solvents for example ethanol, 1-propanol, 2-propanol,
2-methyl-1-propanol and acetone. The Raman spectra of solvates of
benzenesulfonic acid salts of Formula I are shown in FIGS.
5-11.
[0053] While it is possible that, for use in therapy,
therapeutically effective amounts of a compound of Formula I, as
well as anhydrate or hydrate forms thereof, may be administered as
the raw chemical, it is possible to present the active ingredient
as a pharmaceutical composition. Accordingly, the invention further
provides pharmaceutical compositions which include therapeutically
effective amounts of compounds of the Formula I and anhydrate or
hydrate forms thereof, and one or more pharmaceutically acceptable
carriers, diluents, or excipients. The compounds of the Formula I
and anhydrate or hydrate forms thereof, are as described above. The
carrier(s), diluent(s) or excipient(s) must be acceptable in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof. According
to another aspect of the invention there is also provided a process
for the preparation of a pharmaceutical formulation including
admixing a compound of the Formula I, or anhydrate, hydrate or
solvate forms thereof, with one or more pharmaceutically acceptable
carriers, diluents or excipients.
[0054] Compounds of Formula I and anhydrate, hydrate or solvate
forms thereof may be formulated for administration by any route,
and the appropriate route will depend on the disease being treated
as well as the subjects to be treated. Suitable pharmaceutical
formulations include those for oral, rectal, nasal, topical
(including buccal, sub-lingual, and transdermal), vaginal or
parenteral (including intramuscular, sub-cutaneous, intravenous,
and directly into the affected tissue) administration or in a form
suitable for administration by inhalation or insufflation. The
formulations may, where appropriate, be conveniently presented in
discrete dosage units and may be prepared by any of the methods
well know in the pharmacy art.
[0055] Pharmaceutical formulations adapted for oral administration
may be presented as discrete units such as capsules or tablets;
powders or granules; solutions or suspensions in aqueous or
non-aqueous liquids; edible foams or whips; or oil-in-water liquid
emulsions or water-in-oil liquid emulsions.
[0056] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water and the like. Powders are prepared by
comminuting the compound to a suitable fine size and mixing with a
similarly comminuted pharmaceutical carrier such as an edible
carbohydrate, as, for example, starch or mannitol. Flavoring,
preservative, dispersing and coloring agent can also be
present.
[0057] Capsules are made by preparing a powder mixture as described
above, and filling formed gelatin sheaths. Glidants and lubricants
such as colloidal silica, talc, magnesium stearate, calcium
stearate or solid polyethylene glycol can be added to the powder
mixture before the filling operation. A disintegrating or
solubilizing agent such as agar-agar, calcium carbonate or sodium
carbonate can also be added to improve the availability of the
medicament when the capsule is ingested.
[0058] Moreover, when desired or necessary, suitable binders,
lubricants, disintegrating agents and coloring agents can also be
incorporated into the mixture. Suitable binders include starch,
gelatin, natural sugars such as glucose or beta-lactose, corn
sweeteners, natural and synthetic gums such as acacia, tragacanth
or sodium alginate, carboxymethylcellulose, polyethylene glycol,
waxes and the like. Lubricants used in these dosage forms include
sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate,
sodiumacetate, sodium chloride and the like. Disintegrators
include, without limitation, starch, methyl cellulose, agar,
bentonite, xanthan gum and the like. Tablets are formulated, for
example, by preparing a powder mixture, granulating or slugging,
adding a lubricant and disintegrant and pressing into tablets. A
powder mixture is prepared by mixing the compound, suitably
comminuted, with a diluent or base as described above, and
optionally, with a binder such as carboxymethylcellulose, an
aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant
such as paraffin, a resorption accelerator such as a quaternary
salt and/or an absorption agent such as bentonite, kaolin or
dicalcium phosphate. The powder mixture can be granulated by
wetting with a binder such as syrup, starch paste, acadia mucilage
or solutions of cellulosic or polymeric materials and forcing
through a screen. As an alternative to granulating, the powder
mixture can be run through the tablet machine and the result is
imperfectly formed slugs broken into granules. The granules can be
lubricated to prevent sticking to the tablet forming dies by means
of the addition of stearic acid, a stearate salt, talc or mineral
oil. The lubricated mixture is then compressed into tablets. The
compounds of the present invention can also be combined with a free
flowing inert carrier and compressed into tablets directly without
going through the granulating or slugging steps. A clear or opaque
protective coating consisting of a sealing coat of shellac, a
coating of sugar or polymeric material and a polish coating of wax
can be provided. Dyestuffs can be added to these coatings to
distinguish different unit dosages.
[0059] Oral fluids such as solution, syrups and elixirs can be
prepared in dosage unit form so that a given quantity contains a
predetermined amount of the compound. Syrups can be prepared by
dissolving the compound in a suitably flavored aqueous solution,
while elixirs are prepared through the use of a non-toxic alcoholic
vehicle. Suspensions can be formulated by dispersing the compound
in a non-toxic vehicle. Solubilizers and emulsifiers such as
ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol
ethers, preservatives, flavor additive such as peppermint oil or
natural sweeteners or saccharin or other artificial sweeteners, and
the like can also be added.
[0060] Where appropriate, dosage unit formulations for oral
administration can be microencapsulated. The formulation can also
be prepared to prolong or sustain the release as for example by
coating or embedding particulate material in polymers, wax or the
like.
[0061] The compound of Formula I and anhydrate, hydrate or solvate
forms thereof, can also be administered in the form of liposome
delivery systems, such as small unilamellar vesicles, large
unilamellar vesicles and multilamellar vesicles. Liposomes can be
formed from a variety of phospholipids, such as cholesterol,
stearylamine or phosphatidylcholines.
[0062] The compounds of Formula I and anhydrate, hydrate or solvate
forms thereof may also be delivered by the use of monoclonal
antibodies as individual carriers to which the compound molecules
are coupled. The compounds may also be coupled with soluble
polymers as targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropylmethacrylamidephenol,
polyhydroxyethylaspartamide-phenol, or polyethyleneoxidepolylysine
substituted with palmitoyl residues. Furthermore, the compounds may
be coupled to a class of biodegradable polymers useful in achieving
controlled release of a drug, for example, polylactic acid,
polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates
and cross-linked or amphipathic block copolymers of hydrogels.
[0063] Pharmaceutical formulations adapted for transdermal
administration may be presented as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. For example, the active ingredient may
be delivered from the patch by iontophoresis as generally described
in Pharmaceutical Research, 3(6), 318 (1986).
[0064] Pharmaceutical formulations adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, sprays, aerosols or
oils.
[0065] For treatments of the eye or other external tissues, for
example mouth and skin, the formulations are preferably applied as
a topical ointment or cream. When formulated in an ointment, the
active ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water cream base or a
water-in-oil base.
[0066] Pharmaceutical formulations adapted for topical
administrations to the eye include eye drops wherein the active
ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent.
[0067] Pharmaceutical formulations adapted for topical
administration in the mouth include lozenges, pastilles and mouth
washes.
[0068] Pharmaceutical formulations adapted for rectal
administration may be presented as suppositories or as enemas.
[0069] Pharmaceutical formulations adapted for nasal administration
wherein the carrier is a solid include a coarse powder having a
particle size for example in the range 20 to 500 microns which is
administered in the manner in which snuff is taken, i.e. by rapid
inhalation through the nasal passage from a container of the powder
held close up to the nose. Suitable formulations wherein the
carrier is a liquid, for administration as a nasal spray or as
nasal drops, include aqueous or oil solutions of the active
ingredient.
[0070] Pharmaceutical formulations adapted for administration by
inhalation include fine particle dusts or mists, which may be
generated by means of various types of metered, dose pressurised
aerosols, nebulizers or insufflators.
[0071] Pharmaceutical formulations adapted for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams or spray formulations.
[0072] Pharmaceutical formulations adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0073] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations may include other
agents conventional in the art having regard to the type of
formulation in question, for example those suitable for oral
administration may include flavouring agents.
[0074] Also provided in the present invention, is a method for
inhibiting a post proline/analine cleaving protease, such as a
serine protease, such as a dipeptidyl peptidase, such as DPP-IV,
which includes administering a therapeutically effective amount of
a compound of the present invention including anhydrate, hydrate or
solvate forms thereof, to the mammal.
[0075] A therapeutically effective amount of a compound of Formula
I and anhydrate, hydrate or solvate forms thereof will depend on a
number of factors including, but not limited to, the age and weight
of the mammal, the precise disorder requiring treatment and its
severity, the nature of the formulation, and the route of
administration, and will ultimately be at the discretion of the
attendant physician or veterinarian. Typically, the compound of
Formula I and anhydrate, hydrate or solvate forms thereof will be
given for treatment in the range of 0.1 to 100 mg/kg body weight of
recipient (mammal) per day and more usually in the range of 1 to 10
mg/kg body weight per day. Acceptable daily dosages, may be from
about 0.1 to about 1000 mg/day, and preferably from about 0.1 to
about 100 mg/day.
[0076] The compounds of the present invention including anhydrate,
hydrate and/or solvate forms thereof, described above, are useful
in therapy and in the preparation of medicaments for treating a
disorder in a mammal, which is characterized by the need for
inhibition of a post proline/analine cleaving protease, such as a
serine protease, such as a dipeptidyl peptidase, such as DPP IV.
The compounds of the present invention including anhydrate, hydrate
and/or solvate forms thereof are useful for treating or preventing
metabolic disorders, gastrointestinal disorders, viral disorders,
autoimmune disorders, dermatological or mucous membrane disorders,
compliment mediated disorders, inflammatory disorders, and
psychosomatic, depressive, and neuropsychiatric disorders,
including, without limitation, diabetes, obesity, hyperlipidemia,
psoriasis, intestinal distress, constipation, encephalomyelitis,
glumerulonepritis, lipodystrophy, tissue damage, HIV infection,
allergies, inflammation, arthritis, transplant rejection, high
blood pressure, congestive heart failure, tumors, and
stress-induced abortions.
[0077] The compound of Formula I and anhydrate, hydrate or solvate
forms thereof, described above, are useful in therapy and in the
preparation of a medicament for the treatment and/or prophylaxis of
hyperglycemia or other conditions associated with diabetes.
[0078] The following examples are intended for illustration only
and are not intended to limit the scope of the invention in any
way.
EXAMPLE 1
Preparation of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide benzenesulfonate monohydrate
Preparation of trans-4-amino-N,N-dimethylcyclohexanecarboxamide
[0079] To a solution of benzyl
trans-4-[(dimethylamino)-carbonyl]cyclohexylcarbamate (1 wt., 1 eq)
in methanol (7 vol) was added palladium on carbon (0.1 wt). The
reaction was hydrogenated under 0.2 bar over atmosphere of hydrogen
until the reaction was complete (monitored by react IR). After
purging the reaction with nitrogen, the reaction was filtered to
remove the catalyst, and rinsed with methanol (2 vol). The filtrate
was solvent exchanged to acetonitrile (10 vol). This solution of
4-amino-N,N-dimethylcyclohexanecarboxamide was used directly in the
next step.
Preparation of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide
[0080] To a solution of
trans-4-amino-N,N-dimethylcyclohexanecarboxamide (1 wt., 1 eq.) in
acetonitrile (10 vol) was added potassium carbonate (2.3 wt., 2.8
eq.) followed by (2S)-1-(chloroacetyl)-2-pyrrolidinecarbonitrile
(0.96 wt., 0.95 eq.). The resulting slurry was stirred at approx.
25.degree. C. for 20-24 hours. The reaction was filtered, washed
with acetonitrile (1 vol), and the filtrate was concentrated to
minimum volume under vacuum. Dichloromethane (5 vol) and 10%
aqueous citric acid (5 vol) were added and the biphasic mixture was
stirred for 10 minutes. The layers were separated and to the
aqueous layer was added dichloromethane (5 vol) The biphasic
mixture was cooled to approx. 5.degree. C. and 50% aqueous
potassium carbonate (2.3 vol) was added while keeping the
temperature at approx. 5.degree. C. The layers were separated and
the organic layer was washed with brine (2 vol.). The organic layer
(dichloromethane solution of
trans-4-({2-[(2S)-2-cyano-pyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyc-
lohexanecarboxamide) was used directly in the next step.
Preparation of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate monohydrate
[0081] The crude methylene chloride solution from above was charged
to the reaction vessel. Solvent exchange with (8 volumes) of
2-butanone removing excess DCM under reduced pressure while
maintaining the internal temperature at or below 25.degree. C. To
the 2-butanone solution is added 0.11 wt (2 eq) of process water. A
solution of benzenesulfonic acid was prepared by dissolving 0.50 wt
in 2.5 volumes of 2-butanone. Approximately 10% of this solution
was added slowly. Seed crystals were then introduced (0.5 wt %).
The remainder of the acid solution was then added dropwise. After
completion of the addition, the suspension was stirred at high
speed for 1 hr at 25.degree. C. The precipitate was then collected
on Whatman no. 1 filter paper in a Buchner funnel. The cake was
washed successively with 2.times.2 volumes of 2-butanone and then
suction/air dried. The damp cake was then transferred to a
crystallization dish and placed in a vacuum oven (70.degree. C.,
house vacuum, N.sub.2 bleed)
[0082] Expected yield: 80-95%
EXAMPLE 2
Powder X-Ray Diffraction of Monohydrate Besylate Salt
[0083] The monohydrate besylate salt prepared according to Example
1 was prepared by placing the sample on to a silicon zero
background plate and scanning with a conventional Bragg-Brentano
diffractometer with copper K alpha radiation at ambient room
temperature. The powder X-ray diffraction pattern obtained is shown
in FIG. 2.
EXAMPLE 3
Single Crystal X-Ray Diffraction of Monohydrate Besylate Salt
[0084] The single crystal structure of the monohydrate besylate
salt was determined using copper K-alpha radiation at 293K. The
crystal system, space group, and cell parameters are provided:
Crystal System Monoclinic
Space Group: P2.sub.1
Lattice Parameters
[0085] a=11.785(1) .ANG. b=8.367(1) .ANG. c=13.541(1) .ANG.
.beta.=105.657(4) .ANG.
V=1285.7(2) .ANG..sup.3
[0086] The simulated powder X-ray diffraction pattern using copper
K-alpha radiation is shown in FIG. 2a. The differences between the
simulated pattern and the experimental monohydrate pattern in FIG.
2 are primarily due to preferred orientation in the experimental
pattern, minor differences in lattice parameters between single
crystal and experimental powder data, specific peak profile
parameters used in the simulated pattern calculation, and to a
lesser extent due to differences in atomic disorder between the
experimental powder and single crystal data.
EXAMPLE 4
Preparation of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexanecarboxamide besylate anhydrate
[0087] A solution of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexane-carboxamide (1000 mg) in 8 mL of anhydrous methyl
acetate was warmed to 45.degree. C. In a separate vessel, 527 mg (1
eq.) of anhydrous benzene sulfonic acid was dissolved in 2.5 mL of
anhydrous methyl acetate. The solution of benzene sulfonic acid was
then added dropwise to the warm methyl acetate solution described
above. When the addition was complete, the mixture was stirred at
45.degree. C. for an additional 40 minutes and then allowed to cool
naturally to room temperature. The solids were then collected on
Whatman no. 2 filter paper under a N.sub.2 blanket to give 987 mg
(65% th) of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dim
ethylcyclohexanecarboxamide besylate anhydrate.
EXAMPLE 5
Powder X-Ray Diffraction of Anhydrate Besylate Salt
[0088] The anhydrate besylate salt prepared according to Example 4
was prepared by placing the sample on to a silicon zero background
plate and scanning with a conventional Bragg-Brentano
diffractometer with copper K alpha radiation at ambient room
temperature. The powder X-ray diffraction pattern obtained is shown
in FIG. 1.
EXAMPLE 6
Infrared (IR) Studies of
trans-4-({2-[(2S)-2-cyano-pyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyc-
lohexanecarboxamide besylate
[0089] Samples of
trans-4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcycl-
ohexane-carboxamide besylate monohydrate either as a pure solid or
diluted in KBr powder were placed inside a FTIR spectrometer purged
under dry N.sub.2 at room temperature (RT). IR spectra recorded at
different times showed that the intensities of IR bands at 3544.8,
3471.5 and 1626.8 cm.sup.-1, which can be assigned to H.sub.2O,
decreased very rapidly within 10 min. and reached an equilibrium in
.about.30 min. Upon re-exposure of the partially dehydrated samples
to the atmosphere for <1 min., the intensities of these IR bands
are fully recovered. The FTIR spectrum of this experiment is in
FIG. 4.
EXAMPLE 7
Moisture Sorption Testing
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide besylate monohydrate salt
[0090] Moisture sorption was measured with an integrated gas flow
microbalance system. Approximately 20 mg of
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide besylate monohydrate salt was weighed into a sample
pan of an integrated gas flow microbalance system (SGA100, VTI
Corporation). The sample was dried at 60.degree. C. under a dry
nitrogen stream until the rate of weight loss was less than 0.015%
in 5 minutes. After drying the sample was equilibrated at
25.degree. C. and the relative humidity increased stepwise
(adsorption) to 5, 15, 25, 35, 45, 55, 65, 75, 85 and 95% Each
relative humidity step was held until the sample equilibrated at
that condition. Equilibrium was defined as a weight change of less
than 0.015% in 5 minutes. The relative humidity was then decreased
step wise (desorption) to 90, 80, 70, 60, 50, 40, and 20% Each step
was held until equilibrium was reached. The equilibrium condition
was the same as in the sorption phase. The % w/w increase or
decrease in moisture content of the sample is reported for each
equilibrated RH condition.
[0091] Typically, the monohydrate form absorbs less than 10% w/w
water, preferably less than 8% w/w water, more preferably less than
6% w/w water at relative humidity between 0% and 95% at 25.degree.
C. Water adsorbed by monohydrate Form 1 readily desorbs when the
relative humidity is decreased.
EXAMPLE 8
[0092] Moisture sorption testing
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide besylate anhydrate salt
[0093] Moisture sorption was measured with an integrated vacuum
microbalance system. Approximately 11 mg of
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide besylate anhydrate salt was weighed into a sample pan
of an integrated vacuum microbalance system (MB300G, VTI
Corporation). The sample was dried at 60.degree. C. under vacuum
until the rate of weight loss was less than 0.015% in 2 minutes.
After drying the sample was equilibrated at 25.degree. C. and the
relative humidity increased stepwise (adsorption) to 15, 25, 35,
45, 55, 65, 75, 85 and 95% Each relative humidity step was held
until the sample equilibrated at that condition. Equilibrium was
defined as a weight change of less than 0.015% in 2 minutes. The
relative humidity was then decreased step wise (desorption) to 90,
80, 70, 60, 50, 40, 30 and 20% Each step was held until equilibrium
was reached. The equilibrium condition was the same as in the
sorption phase. The % w/w increase or decrease in moisture content
of the sample is reported for each equilibrated RH condition.
[0094] Typically, the anhydrate form converts to the hydrate
between 65% and 75% relative humidity at 25.degree. C.
EXAMPLE 9
Raman Spectra of
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide solvates In a typical process,
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide is suspended in ethanol in the presence of seed
material and warmed to 60.degree. C. for one hour. All Raman
spectra were obtained on a Thermo Nicolet 960 FT Raman spectrometer
equipped with a ViewStage with a spectral resolution of 4
cm.sup.-1. FT Raman Spectra are given in FIGS. 5-11.
[0095] Below is a table summarizing several of the major peaks and
their corresponding relative intensities. As noted hereinabove, due
to the variability appreciated by those skilled in the art, the
existence of peaks and intensities should not necessarily be
interpreted too critically in the characterization of a compound
within the scope of the present invention.
TABLE-US-00005 Raman Peak Table of Formula I Solvates 2-methyl-1-
ace- Form 1 Form 2 Ethanol 1-propanol propanol 2-propanol tone 96
93 109 99 106 98 108 135 106 139 139 139 143 156 317 136 294 305
287 304 312 375 155 322 318 317 318 370 515 312 375 370 368 370 415
618 368 451 447 491 447 452 644 450 481 463 515 462 516 727 517 517
482 574 485 568 793 618 570 515 616 515 617 809 638 618 554 642 572
639 846 717 639 573 728 615 718 997 726 730 616 793 644 727 1018
793 793 644 810 728 793 1035 832 834 729 832 792 832 1122 870 881
792 874 820 870 1163 925 929 832 929 831 927 1246 993 941 855 998
872 998 1269 1000 973 929 1014 928 1033 1316 1018 997 943 1033 943
1071 1351 1032 1019 973 1072 996 1126 1443 1072 1036 997 1125 1013
1159 1482 1125 1129 1014 1158 1033 1174 1587 1143 1163 1034 1174
1074 1227 1622 1166 1251 1074 1265 1123 1259 1680 1227 1269 1124
1295 1157 1314 2240 1246 1291 1158 1342 1243 1359 2871 1261 1338
1268 1365 1268 1421 2891 1312 1366 1295 1423 1342 1443 2956 1357
1420 1343 1447 1364 1454 3003 1422 1450 1365 1588 1423 1588 3068
1442 1486 1423 1624 1445 1620 1454 1588 1445 1675 1587 1662 1587
1635 1588 2244 1620 1712 1641 1681 1622 2717 1674 2240 1664 2240
1675 2871 2246 2867 2242 2870 2246 2935 2878 2888 2864 2919 2877
2961 2929 2935 2899 2949 2961 3065 2962 2952 2947 2981 2988 2989
2968 2972 2994 3063 3061 2984 2982 3054 3003 3003 3066 3016 3060
3062
EXAMPLE 10
[0096] Powder X-Ray Diffraction of
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide hydrochloride salt
[0097] The powder X-ray diffraction pattern for
4-({2-[(2S)-2-cyanopyrrolidinyl]-2-oxoethyl}amino)-N,N-dimethylcyclohexan-
ecarboxamide hydrochloride salt is shown in FIG. 12.
[0098] Below is a table summarizing the major peaks and their
corresponding relative intensities.
TABLE-US-00006 No. 2Theta d I (cps) I/Io FWHM 1 6.4 13.7 1243 940
0.16 2 9.7 9.1 110 65 0.16 3 12.9 6.8 177 120 0.16 4 15.6 5.7 534
375 0.18 5 15.9 5.6 376 245 0.14 6 16.6 5.3 380 233 0.20 7 17.2 5.2
1204 874 0.20 8 17.8 5.0 324 178 0.16 9 18.1 4.9 448 275 0.18 10
18.7 4.7 241 109 0.16 11 19.1 4.6 393 227 0.16 12 19.5 4.6 482 301
0.16 13 20.0 4.4 914 652 0.18 14 20.7 4.3 196 110 0.16 15 22.2 4.0
741 518 0.20 16 22.8 3.9 1366 1000 0.18 17 23.0 3.9 453 279 0.06 18
23.8 3.7 522 330 0.20 19 24.7 3.6 442 275 0.22 20 25.4 3.5 214 108
0.22 21 27.5 3.2 155 62 0.38 22 28.2 3.2 403 249 0.18 23 28.5 3.1
438 276 0.16 24 28.8 3.1 176 71 0.08 25 29.8 3.0 266 153 0.28 26
30.3 2.9 123 50 0.24 27 32.0 2.8 258 142 0.22 28 32.3 2.8 360 222
0.20 29 32.8 2.7 190 89 0.44 30 34.0 2.6 123 47 0.28 31 34.8 2.6
134 60 0.22 32 35.1 2.6 125 50 0.06 33 35.3 2.5 137 56 0.18 34 36.2
2.5 145 59 0.20 35 36.9 2.4 157 74 0.24
Biological Data
Materials:
[0099] H-Ala-Pro-pNA*HCl was purchased from BACHEM Bioscience Inc.
(product no. L-1115). A 500 mM stock solution was prepared with
dimethylsulfoxide and stored at -20.degree. C. Gly-Pro-AMC was
purchased from Enzyme System Products (product no. AMC-39) and
stored at -20.degree. C. as a 10 mM stock solution in
dimethylsulfoxide. Test compound was dissolved to 10 mM in
dimethylsulfoxide and this was used as a stock solution for DPP-IV
titration assay. Athens Research and Technology, Inc prepared the
purified human DPP-IV. The material was isolated from human
prostasomes using the method of DeMeester et al., J. Immunol.
Methods 189, 99-105. (1996), incorporated herein by reference to
the extent of describing such method.
DPP-IV Assay:
[0100] Two-fold serial dilutions of test compounds in 100%
dimethylsulfoxide were performed in 96-well polystyrene flat bottom
plates (Costar, #9017). The average enzymatic activity from wells
containing dimethylsulfoxide but lacking test compound was used as
a control value for calculating percent inhibition. DPP-IV (20
ng/mL) was mixed in microtiter plates with test compound, substrate
and assay buffer to yield 100 .mu.M H-Ala-Pro-pNA.HCl in 25 mM
Tris, pH 7.5, 10 mM KCl, 140 mM NaCl. The intact peptide contains a
p-nitrophenylanilide which, when hydrolyzed by DPP-IV, releases the
absorbent p-nitrophenylaniline. The absorbency was monitored in 20
minutes intervals at a wavelength of 387 nm using a Molecular
Devices SpectraMax 250 absorbency plate reader. The enzymatic
activity was determined by estimating the best linear fit to the
data. Values for enzymatic activity were taken directly from the
linear fit determined by the software on the plate reader.
Data Analysis The enzymatic activity was determined by estimating
the best linear fit to the data. Data reduction was performed using
the Microsoft Excel RoboSage.
[0101] Determination of IC.sub.50 values: The enzymatic activity
was plotted against the concentration of test compound, including
[I]=0, and the IC.sub.50 determined from a fit of equation (2) to
the data.
RATE=V.sub.max/(1+([I]/IC.sub.50)) (2)
[0102] V.sub.max was the best fit estimate of the maximal enzymatic
activity.
[0103] Determination of K.sub.i values: K.sub.i values were
calculated from IC.sub.50 values using equation (3) assuming a
competitive model.
K i = I C 50 * [ 1 - S ( S + K m ) ] ( 3 ) ##EQU00001##
[0104] The apparent pKi values for the test compound was
>5.0.
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